Infection with influenza virus causes a highly contagious disease of the respiratory tract. Influenza represents a unique therapeutic challenge due to both seasonal antigen shift and drift. Conventional approaches for therapeutic targeting of influenza pathogens have consistently faced obstacles arising from the development of resistant strains and a lack of broad-spectrum application. The overall goal of this project is to develop broad- spectrum Host Oriented Therapeutic antibodies that are active against all strains of influenza including those that are resistant to current antiviral therapies. Phase I of this project has been successfully completed. Our original proposal centered upon a newly- developed approach, Random Homozygous Gene Perturbation (RHGP), which can simultaneously knockdown both copies of a target gene and identify those target knock-outs that are well-tolerated in normal cells but inhibit influenza infection. We applied RHGP to identify a set of host-oriented targets that prevent influenza killing of host cells and validate these targets. We further emphasized one particular pathway, based on the RHGP discovery of Nedd4 Binding Protein 2 (N4BP2) and WWP2, a member of Nedd4 family. This investigation allowed us to demonstrate that Nedd4 is exposed on the surface of influenza-infected cells but not on non-infected cells. This unique discovery positions us with an opportunity to develop a broad-spectrum inhibitor of influenza infection, which could act both as a therapeutic and prophylactic therapy to combat seasonal or pandemic forms of influenza. The specific aims of Phase II are to 1) Create human scFv libraries and screen the candidate antibodies using Nedd4 protein and the peptides. We will isolate approximately 200 different Nedd4-specific scFvs, from which we will select at least 10 different candidates that demonstrate immunoreactivity against a broad-spectrum of seasonal influenza variants and other potential pandemic variants. The leading ten scFv candidates will then be constructed into full-length human IgG1. 2) Evaluate the safety and antiviral activity of the antibody candidates to identify a lead candidate for IND-enabling studies through ADCC, CDC and direct antiviral activity. 3) Optimize the lead candidate to enable the nomination of a Clinical Candidate that has the greatest potential for becoming a successful IND. We believe the concept of selective targeting of influenza-infected cells has exciting prospects and that it is feasible to develop a therapeutic monoclonal antibody that is applicable to a broad spectrum of seasonal and pandemic influenza strains. PUBLIC HEALTH RELEVANCE: Conventional approaches for therapeutic targeting of the influenza virus have consistently faced obstacles arising from a high viral mutation rate and a lack of broad-spectrum application. The overall goal of this project is to develop broad-spectrum Host Oriented Therapeutic antibodies that target Nedd4, a molecule that is selectively exposed on the surface of influenza-infected cells. These antibodies are expected to be active against all strains of influenza, including those with resistance to current antiviral therapies.